Isolation and Whole-Genome Sequencing of Four Antibiotic-Producing Pseudomonas Strains

ABSTRACT Here, we report the isolation, whole-genome sequencing, and annotation of four novel Pseudomonas isolates. We also evaluate the biosynthetic potential of each genome.

P seudomonas is a Gram-negative, rod-shaped, polar flagellated bacterial genus with more than 140 identified species (1). Within this genus, there are many species that inhabit a diverse range of environments, resulting in their multifaceted metabolic capacities and adaptation abilities in changing environments. Here, we report the draft genomes of four novel Pseudomonas isolates that exhibit high antibiotic activity.
Soil samples were collected from a variety of locations (Table 1). One gram of each sample was resuspended in 10 mL of sterile saline, diluted serially, and plated on Reasoner's 2 agar (R2A) (2) hardened medium plates at 25°C for 48 h. Once diluted, 50 colonies were isolated, and individual colonies were screened for antibiotic activity by patching onto lawns of other bacteria, followed by incubation at 25°C for 48 h. The strains described here, LF19, LM20, JR33AA, and KCA11, were selected because they produced zones of inhibition against lawns of several bacterial species (Table 1). The 16S rRNA gene PCR product was sequenced from each strain using the primer set 27F (59-AGR GTT TGA TYM TGG CTC AG-39) and 1492R (59-GGY TAC CTT GTT ACG ACT T-39), with 55°C annealing and 30 s of extension. Using NCBI BLAST (3), it was determined that the isolates belong to the Pseudomonas genus.
Axenic cultures grown on R2A were sent to the Microbial Genome Sequencing Center (Pittsburgh, PA) for DNA isolation using the Qiagen DNeasy blood and tissue kit and wholegenome sequencing (Fig. 1A). Sequence libraries were prepared with a small-volume tagmentation protocol using the Nextera DNA library preparation kit (Illumina, San Diego, CA, USA). Barcodes and adapters were attached, and libraries were amplified using the KAPA HiFi library amplification kit (4). Paired-end libraries were subsequently sequenced on the Illumina NextSeq 550 platform. FastQC v0.11.9 (5, 6) was used to verify the quality of the reads. These reads were then assembled de novo in PATRIC v3.6.12 (7) using the Unicycler v0.4.8 program (8). The assembly included polishing using two Pilon iterations (9) and examination using QUAST v5.0.2 (10). Read number and length information can be found in Table 1. All programs related to genome assembly were run with default parameters. Annotation was performed in PATRIC v3.6.12 using the RASTtk pipeline (11) with default bacterial parameters, using Pseudomonas as a taxonomic guide (Table 1).
Taxonomic relationships between the four isolates and other known Pseudomonas species were evaluated using the Codon Tree pipeline (12-15) in PATRIC v3.6.12 (7) in combination with average nucleotide identity (ANI) calculations using the Kostas Lab

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Microbiology Resource Announcements ANI Calculator v1.0 (16,17), both run with default parameters. These calculations revealed that LM20 and KCA11 shared 99.5% nucleotide identity, while the remaining relationships all ranged between 82.5% and 89.3%. Additionally, isolates LM20, KCA11, and JR33AA all group with Pseudomonas putida strains, while LF19 is most related to Pseudomonas wadenswilerensis and Pseudomonas donghuensis strains (Fig. 1B). Genome mining for antibacterial compounds using antiSMASH v6.0.1 (18), with the relaxed strictness setting, provided evidence that each of the four strains contains biosynthetic gene clusters (BGCs) potentially encoding secondary metabolites. The strain with the most predicted BGCs is LF19, with 13, and the strain with the least is LM20, with 8. KCA11 and JR33AA are both predicted to possess 10 BGCs. These strains add to the genomic data available for the Pseudomonas genus, supporting further investigation into its biosynthetic potential.
Data availability. This whole-genome shotgun project was deposited in GenBank under BioProject PRJNA784595. GenBank assembly and Sequence Read Archive (SRA) accession numbers are presented in Table 1.